1. Field of the Invention
The invention is generally related to the fields of pharmacology and immunology. More specifically, the invention is directed to novel compositions of matter for selectively activating T cells, and having reduced activation of Endothelial cells or fibroblasts. The novel compositions include variants of the cytokine family, and in particular human Interleukin-4 (IL-4).
2. Description of Related Art
Interleukin 4 (IL-4) is a pleiotropic cytokine, having activities on cells of the immune system, endothelium, and those of fibroblastic nature. Reported in vitro effects of IL-4 administration include proliferation of B cells, immnunoglobulin class switching in B cells. In T cells, IL-4 stimulates T cell proliferation after preactivation with mitogens and down-regulates IFN-xcex3 production. In monocytes, IL-4 induces class II MHC molecules expression, release of lipopolysaccharide-induced tPA, and CD23 expression. In Endothelial cells (EC), IL-4 induces expression of VCAM-1 and IL-6 release, and decreases ICAM-1 expression. (Maher, D W, et al., Human Interleukin-4: An Immunomodulator with Potential Therapeutic Applications, Progress in Growth Factor Research, 3:43-56 (1991)).
Because of its ability to stimulate proliferation of T cells activated by exposure to IL-2, IL-4 therapy has been pursued. For instance, IL-4 has demonstrated anti-neoplastic activity in animal models of renal carcinomas, and has induced tumor regression in mice (Bosco, M., et al., Low Doses of IL-4 Injected Perilymphatically in Tumor-bearing Mice Inhibit the Growth of Poorly and Apparently Nonimmunogenic Tumors and Induce a Tumor Specific Immune Memory, J. Immunol., 145:3136-43 (1990)). However, its toxicity limits dosage in humans (Margolin, K, et al., Phase II Studies of Human Recombinant Interleukin-4 in Advanced Renal Cancer and Malignant Melanoma, J. Immunotherapy, 15:147-153 (1994)).
Because of its immunoregulatory activity, a number of clinical applications are suggested for IL-4. Among these clinical applications are disorders caused by imbalances of the immune system, particulary those caused by imbalances of T helper (Th) cell responses to antigen. These diseases include certain autoimmune diseases, rheumatic diseases, dermatological diseases, and infectious diseases. A large body of experimental work has established that Th cells fall into two broad classes, designated Th1 and Th2 (Mosmann, T. R., Cherwinski, H., Bond, M. W., Giedlin, M A. and Coffinan, R. L., Two types of murine helper T cell clone. I. Definition according to profiles of lymphokine activities and secreted proteins, J. Immunol., 136:2348-2357 (1986); Mosmann, T. R., Cytokines, differentiation and functions of subsets of CD4 and CD8 T cells, Behring Inst. Mitt., 1-6 (1995)). These T cell classes are defined by the cytokines they express: Th1 cells make IL-2, INF-xcex3, and TNF-xcex1, while Th2 cells make IL-4 and IL-5. Th1 and Th2 cells are formed from naive CD4+T cells. Differentiation into Th1 or Th2 subsets depends on the cytokine present during antigen stimulation: IFN-xcex3 and IL-12 direct differentiation of naive cells to the Th1 phenotype, while IL-4 directs differentiation to the Th2 phenotype. While the Th1 and Th2 subsets may represent extremes along a continuum of Th cell phenotypes (for example, ThO cells, which express low levels of both INF-xcex3 and IL-4, have been described), this classification nevertheless is the major paradigm in the field of immunology for describing the character of the immune response.
It has been observed that certain organ-specific autoimmune diseases are associated with a predominantly Th1 T cell response against autoantigen (Liblau R S; Singer S M;, McDevitt H O, Th1 and Th2 CD4+ T cells in the pathogenesis of organ-specific autoimmune diseases, Immunol. Today, 16:34-38 (1995)). One such autoimmune disease is insulin-dependent diabetes (IDDM), a disorder characterized by T cell-mediated destruction of pancreatic xcex2 cells. Several lines of evidence suggest that Th1-type cells are primarily responsible for the pancreatic xcex2 cell destruction (reviewed in Tisch, R. et al., Review: Insulin-dependent Diabetes Mellitus, Cell, 85:291-297 (1996)). Administration of IL-4 to NOD mice, which serves as an animal model of IDDM, down-regulates the Th1 cell population and significantly delays the onset of diabetes (Rapoport, et al., IL-4 Reverses T cell Proliferation Unresponsiveness and Prevents the Onset of Diabetes in NOD Mice, J. Exp. Med., 178:87-99 (1993)). Another such autoimmune disease is multiple sclerosis (MS), a disease which is characterized by an autoimmune attack upon the myelin sheath surrounding nerve cells. Studies in humans with MS have demonstrated that exacerbation of MS is associated with the presence of autoantigen-specific Th1 and Th0 cells and that remission is associated with the presence of autoantigen-specific Th2 and Th0 cells (Correale, J et al., Patterns of cytokine secretion by autoreactive proteolipid protein-specific T cell clones during the course of multiple sclerosis, J. Immunol., 154:2959-2968 (1995)). Mice with experimental autoimmune encephalomyelitis (EAE), an animal model for MS, also exhibit the Th1 cell polarization (Cua, D J, Hinton, D R, and Stohlman, S A, J. Immunol., 155:4052-4059 (1995)). Indirect evidence from a study in the EAE model suggests that IL-4 plays a critical role in disease attenuation resulting from treatment with a tolerogenic peptide (Brocke, S. et al. Treatment of experimental encephalomyelitis with a peptide analogue of myelin basic protein, Nature, 379:343-346 (1996)).
Other autoimmune diseases such as Rheumatoid Arthritis (RA) are also targets for IL-4 based therapies. Animal models of RA have shown a disequilibrium of cell profiles tilting towards Th1 cells, and in mice that overexpress TNF-xcex1, anti-TNF-xcex1 antibodies have demonstrated disease attenuation, suggesting that IL-4 therapies that result in down-regulation of Th1 cell populations may have an anti-TNF-xcex1 effect also. (See Feldmann, M, et al., Review: Rheumatoid Arthritis, Cell, 85:307-310 (1996)).
Psoriasis vulgaris is a chronic dermatologic disorder characterized by infiltration of affected skin with monocytes and T cells. Several reports indicate that psoriatic skin lesional T cells and PBL are predominantly of the Th1 phenotype (Uyemura K; Yamamura M; Fivenson D F; Modlin R L; Nickoloff B J, The cytokine network in lesional and lesion-free psoriatic skin is characterized by a T-helper type 1 cell-mediated response, J Invest Dermatol., 101:701-705 (1993); Schlaak J F; Buslau M; Jochum W, Hermann E; Girndt M; Gallati H; Meyer zum Buschenfelde K H; Fleischer B, T cells involved in psoriasis vulgaris belong to the Th1 subset, J Invest Dermatol, 102:145-149 (1994)). Furthermore, monomethylfumarate, a drug which has been reported to be of clinical benefit to patients with psoriasis, has been shown to selectively stimulate Th2 cytokine secretion from PBMC (de Jong R; Bezemer A C; Zomerdijk T P; van de Pouw-Kraan T, Ottenhoff T H; Nibbering P H, Selective stimulation of T helper 2 cytokine responses by the anti-psoriasis agent monomethylfumarate, Eur J Immunol, 26:2067-2074 (1996)). Therefore, IL-4 would be expected to reverse the Th polarization and be of clinical benefit in psoriasis.
Certain infectious diseases are associated with polarized Th cell responses to the infectious agent. Th2 responses have in some cases been associated with resistance to the infectious agent. An example is Borrelia burgdorfei, the infectious agent for Lyme disease. Humans infected with B. burgdorferi exhibit a predominantly Th1 -like cytokine profile (Oksi J; Savolainen J; Pene J; Bousquet J; Laippala P; Viijanen M K, Decreased interleukin-4 and increased gamma interferon production by peripheral blood mononuclear cells of patients with Lyme borreliosis, Infect. Immun., 64:3620-3623 (1996)). In a mouse model of B. burgdoreri-induced arthritis, resistance to disease is associated with IL-4 production while susceptibility is associated with INF-xcex3 production (Matyniak J E; Reiner S L, T helper phenotype and genetic susceptibility in experimental Lyme disease, J Exp Med, 181(3):1251-1254 (1995); Keane-Afyers A; Nickell S P, Role of IL-4 and IFN-gamma in modulation of immunity to Borrelia burgdorferi in mice, J Immunol, 155:2020-2028 (1995)). Treatment of B. burgdorferi-infected mice with IL-4 augments resistance to the infection (Keane-Myers A; Maliszewski C R; Finkelman F D; Nickell S P, Recombinant IL-4 treatment augments resistance to Borrelia burgdorferi infections in both normal susceptible and antibody-deficient susceptible mice, J Immunol., 156:2488-2494(1996)).
IL-4 has been reported to have a direct effect on inhibiting the growth of lymphomas and leukemias (Akashi, K, The role of interleukin-4 in the negative regulation of leukemia cell growth, Leuk Lymphoma, 9:205-9 (1993)). For example, IL-4 has been reported to induce apoptosis in cells from patients with acute lymphoblastic leukemia (Manabe, A, et al., Interleukin-4 induces programmed cell death (apoptosis) in cases of high-risk acute lymphoblastic leukemia, Blood, 83:1731-7 (1994)), and inhibits the growth of cells from patients with non-Hodgkin""s B cell lymphoma (Defrance, T, et al., Antiproliferative effects of interleukin-4 on freshly isolated non-Hodgkin malignant B-lymphoma cells, Blood, 79:990-6 (1992)).
IL-4 has also been reported to exhibit activities which suggests that it would be of clinical benefit in osteoarthritis. Osteoarthritis is a disease in which the degradation of cartilage is the primary pathology (Sack, K E, Osteoarthritis, A continuing challenge, West J Med, 163:579-86 (1995); Oddis, C V, New perspectives on osteoarthritis, Am J Med, 100:10S-15S (1996)). IL-4 inhibits TNF-xcex1 and IL-1 beta production by monocytes and synoviocytes from osteoarthritic patients (Bendrups, A, Hilton, A, Meager, A and Hamilton, J A, Reduction of tumor necrosis factor alpha and interleukin-1 beta levels in human synovial tissue by interleukin-4 and glucocorticoid, Rheumatol Int, 12:217-20 (1993); Seitz, M, et al., Production of interleukin-1 receptor antagonist, inflammatory chemotactic proteins, and prostaglandin E by rheumatoid and osteoarthritic synoviocytesxe2x80x94regulation by IFN-gamma and IL-4, J Immunol, 152:2060-5 (1994)). Additionally, IL-4 has been reported to directly block the degradation of cartilage in ex vivo cartilage explants (Yeh, L A, Augustine, A J, Lee, P, Riviere, L R and Sheldon, A, Interleukin-4, an inhibitor of cartilage breakdown in bovine articular cartilage explants, J Rheumatol, 22:1740-6 (1995)). These activities suggest that IL-4 would be of clinical benefit in osteoarthritis.
However, the clinical use of IL-4 has been limited due to its acute toxicity, which is manifested as a vascular leak syndrome (Margolin, K, et al., Phase II Studies of Human Recombinant Interleukin-4 in Advanced Renal Cancer and Malignant Melanoma, J. Immunotherapy, 15:147-153 (1994)). There is no art in the literature which describes the mechanism of the acute toxic effect of IL-4, nor that describes analogs or mutants of IL-4 that retain immunoregulatory activities but have reduced acute toxicity.
IL-4 mutant proteins (xe2x80x9cmuteinsxe2x80x9d) are known. The IL-4 mutein IL-4/Y 124D is a T cell antagonist (Kruse N, Tony H P, Sebald W, Conversion of human interleukin-4 into a high affinity antagonist by a single amino acid replacement, Embo J, 11:3237-44 (1992)).
Therapeutic uses of IL-4 found in patents or patent applications include the following: the use of IL-4 for potentiation of anticancer effects of chemotherapeutic agents, particularly Hodgkin""s Disease and non-Hodgkins Lymphoma (see WO 9607422); the use of antigenic fragments of IL-4 to generate antibodies to treat IL-4 related diseases by suppressing or imitating the binding activity of IL-4 (see WO 9524481), and to detect, measure and immunopurify IL-4 (see WO 9317106); for inducing the differentiation of precursor B cells to Immunoglobulin secreting cells, the mature B cells being useful for restoring immune function in immune-compromised patients (see WO 9404658); when used in combination with IL-10, as a therapy for treatment of leukemia, lymphoma, inflammatory bowel disease and delayed type hypersensitivity (e.g. ulcerative colitis and Crohn""s Disease) (see WO 9404180); treatment of HIV infection by administering IL-4 to inhibit viral replication in monocytes and macrophages, and to increase their cytotoxicity towards some tumor cells (see WO 9404179); for stimulation of skin fibroblast proliferation for treating wounds in diabetic and immuno-compromised patients (see WO 9211861); for enhancing the primary immune response when administering bacterial, toxoid, and viral vaccines, especially tetanus toxoid vaccine (see WO 9211030); for inhibition of IL-2 induced proliferation of B cell malignancies, especially chronic lymphocytic leukemia, non-Hodgkin""s malignant lymphoma (see WO 9210201); use of IL-4 to treat melanomas, renal and basal cell carcinomas (see WO 9204044).
The patent literature discloses IL-4 proteins and some muteins, but none directed to an IL-4 therapy with reduced side effects. Lee et al. U.S. Pat. No. 5,017,691 (xe2x80x9cthe ""691 patentxe2x80x9d) is directed to mammalian proteins and muteins of human IL-4 which disclose both B-cell growth factor activity and T cell growth factor activity. It discloses nucleic acids coding for polypeptides exhibiting IL-4 activity, as well as the polypeptides themselves and methods for their production. Muteins to the wild-type IL-4 at amino acid positions are disclosed that retain their ability to stimulate both B- and T cell proliferation in vitro. However, nothing in Lee suggests any T cell selective IL-4 muteins, anticipated activation of EC""s or the endothelial cell leakiness which accompanies administration of IL-4. Thus, IL-4 itself is not enabling as a therapeutic modality because of the dose-limiting toxicity.
U.S. Pat. No. 5,013,824 describes hIL-4 peptide derivatives comprising from 6 to 40 amino acids of the native hIL-4. Also disclosed are immunogens comprising conjugates of the peptides and carriers. Carriers include erythrocytes, bacteriophages, proteins, synthetic particles or any substance capable of eliciting antibody production against the conjugated peptide. No muteins of IL-4 are disclosed.
WO96/04306-A2 discloses single-muteins that are antagonists and partial agonists of hIL-2 and hIL-13. No data regarding IL-4 is disclosed. WO95/27052 discloses splice mutants of IL-2 and IL-4 containing exons 1, 2 and 4.
There exists a need for an improved IL-4 molecule which has reduced toxicity and is more generally tolerated.
The invention is directed to human IL-4 muteins numbered in accordance with wild-type IL-4 having T cell activating activity, but having reduced endothelial cell activating activity. In particular, human IL-4 muteins wherein the surface-exposed residues of the D helix of the wild-type IL-4 are mutated whereby the resulting mutein causes T cell proliferation, and causes reduced IL-6 secretion from HUVECs, relative to wild-type IL-4. This invention realizes a less toxic IL-4 mutein that allows greater therapeutic use of this interleukin.
Further, the invention is directed to IL-4 muteins having single, double and triple mutations represented by the designators R121A, R121D, R121E, R121F, R121H, R121I, R121K, R121N, R121P, R121T, R121W; Y124A, Y124Q, Y124R, Y124S, Y124T; Y124A/S125A, T13D/R121E; and R121T/E122F/Y124Q, when numbered in accordance with wild type IL-4 (His=1). The invention also includes polynucleotides coding for the muteins of the invention, vectors containing the polynucleotides, transformed host cells, pharmaceutical compositions comprising the muteins, and therapeutic methods of treatment.
The invention is also directed to a vector comprising the polynucleotide encoding a mutein of this invention, the vector directing the expression of a human IL-4 mutein having T cell activating activity but having reduced endothelial cell activating activity, the vector being capable of enabling transfection of a target organism and subsequent in vivo expression of said human IL-4 mutein coded for by said polynucleotide.
The invention is also directed to a method of selecting a human IL-4 mutein numbered in accordance with wild-type IL-4 having T cell activating activity but having reduced endothelial cell activating activity, comprising mutating the surface-exposed residues of the D helix of the wild-type IL-4 whereby the resulting mutein causes T cell proliferation, and causes reduced IL-6 secretion from HUVECs, relative to wild-type.
The invention is also directed to a method of treating a patient afflicted with an IL-4 treatable condition by administering a therapeutically effective amount of a human IL-4 mutein numbered in accordance with wild-type IL-4 having T cell activating activity but having reduced endothelial cell activating activity. This method is applicable wherein the IL-4 treatable condition is an autoimmune disorder, cancer, infectious disease, cartilage disorder, and psoriatic disorders.